Clin Genet 2011
Printed in Singapore. All rights reserved
© 2011 John Wiley & Sons A/S
Cystic fibrosis mutations for p.F508del
compound heterozygotes predict sweat
chloride levels and pancreatic sufficiency
Sebro R, Levy H, Schneck K, Dimmock D, Raby BA, Cannon CL,
Broeckel U, Risch NJ. Cystic fibrosis mutations for p.F508del compound
heterozygotes predict sweat chloride levels and pancreatic sufficiency.
Clin Genet 2011. © John Wiley & Sons A/S, 2011
Cystic fibrosis (CF) is a monogenetic disease with a complex phenotype.
Over 1500 mutations in the CFTR gene have been identified; however, the
p.F508del mutation is most common. There has been limited correlation
between the CFTR mutation genotype and the disease phenotypes. We
evaluated the non-p.F508del mutation of 108 p.F508del compound
heterozygotes using the biological classification method, Grantham and
Sorting Intolerant from Tolerant (SIFT) scores to assess whether these
scoring systems correlated with sweat chloride levels, pancreatic
sufficiency, predicted FEV1, and risk of infection with Pseudomonas
aeruginosa in the last year. Mutations predicted to be ‘mild’ by the
biological classification method are associated with more normal sweat
chloride levels (p < 0.001), pancreatic sufficiency (p < 0.001) and
decreased risk of infection with Pseudomonas in the last year (p = 0.014).
Lower Grantham scores are associated with more normal sweat chloride
levels (p < 0.001), and pancreatic sufficiency (p = 0.014). Higher SIFT
scores are associated with more normal sweat chloride levels (p < 0.001)
and pancreatic sufficiency (p = 0.011). There was no association between
pulmonary function measured by predicted FEV1and the biological
classification (p = 0.98), Grantham (p = 0.28) or SIFT scores (p = 0.62),
which suggests the pulmonary disease related to CF may involve other
modifier genes and environmental factors.
Conflict of interest
Nothing to declare.
R Sebroa,b,∗, H Levyc,d,e,∗,
K Schneckd,e, D Dimmockf,
BA Rabyg, CL Cannonh,
U Broeckeliand NJ Rischa,j,k
aInstitute for Human Genetics, and
bDepartment of Radiology and
Biomedical Imaging, University of
California, San Francisco, San Francisco,
CA, USA,cDivision of Pulmonary and
Sleep Medicine, anddClinical Research
Institute of the Children’s Hospital of
Wisconsin, Milwaukee, WI, USA,
eDepartment of Pediatrics, Medical
College of Wisconsin, Milwaukee, WI,
USA,fDivision of Genetics, Children’s
Hospital of Wisconsin, Milwaukee, WI,
USA,gChanning Laboratory, Brigham
and Women’s Hospital, Boston, MA,
USA,hDepartment of Pediatrics, UT
Southwestern Medical Center, Dallas,
Texas, USA,iThe Human and Molecular
Genetics Center, The Medical College of
Wisconsin, Milwaukee, WI, USA,
jDepartment of Epidemiology and
Biostatistics, University of California, San
Francisco, USA, and
Research, Kaiser, Oakland, CA, USA
∗Both authors contributed equally to this
Key words: cystic fibrosis – Grantham –
pulmonary function – SIFT – sweat
Corresponding author: Ronnie Sebro,
MD, PhD, Department of Radiology and
Biomedical Imaging, 505 Parnassus
Ave, M-391, University of California,
San Francisco, San Francisco, CA
Tel.: +1 415 476 1127;
fax: +1 415 476 1356;
Received 3 September 2011, revised
and accepted for publication 25
Sebro et al.
Cystic fibrosis (CF) is a Mendelian autosomal reces-
sive disease that shows phenotypic heterogeneity and
can manifest as chronic sinopulmonary disease, pan-
creatic insufficiency, elevated sodium chloride loss in
sweat, male infertility and gastrointestinal abnormali-
ties, including liver disease (Fig. 1). Pulmonary disease
is the major cause of mortality in CF subjects (1) and
most clinically relevant phenotype. Over 1500 unique
mutations have been identified in the CFTR gene (2).
The most common allele, p.F508del, has a frequency
of over 66% in Caucasians (3). Other mutations have
a frequency of less than 5%, although some can be
more prevalent in some subpopulations because of
founder effects. Because of the vast number of muta-
tions, genetic testing is limited for definitive diagnosis,
and the sweat chloride test remains pivotal for diagno-
sis. Usually, sweat chloride levels above 60 mmol/l are
diagnostic (4). There have been multiple efforts to cor-
relate CFTR mutation with phenotype to understand the
specific effect of each mutation. The biological classifi-
cation score is based on in vitro mutation analysis and
is the most accepted classification scheme. Mutations
are classified based on known molecular consequences:
class I – no synthesis; class II – processing blocked;
class III – regulation blocked; class IV – altered con-
ductance; and class V – reduced synthesis (5). Muta-
tions in a higher class are thought to be milder and
some class IV and class V mutations have been asso-
ciated with pancreatic sufficiency (6). Data support a
probable correlation between the CFTR mutation and
Pseudomonas infection (7). Missense and splice site
mutations that are considered mild CF allelic variants
have also been associated with lower risk for acquisition
of Pseudomonas (8).
We analyzed whether genotype–phenotype correla-
tions exist between CFTR mutations and four clinical
phenotypes – sweat chloride levels, pancreatic suffi-
ciency, pulmonary function measured by the predicted
forced expiratory volume in 1 s (predicted FEV1),
and probability of infection with Pseudomonas in the
last year. We consider three mutation scoring algo-
rithms – the biological classification, Grantham and
Sorting Intolerant from Tolerant (SIFT) scores and
Meconium, ileus, DIOS
Fig. 1. Factors that may affect the correlation between CFTR genotype
assess whether these scores correlate with the disease
Materials and methods
We reviewed clinical data from 435 CF subjects fol-
lowed at Children’s Hospital, Boston (CHB). Inclu-
sion criteria required subjects to have two copies of
a known CFTR mutation or elevated sweat chloride
levels (>60 mmol/l). FEV1was measured by standard
spirometry according to American Thoracic Society cri-
teria, and absolute values were converted to a percent-
age of the predicted volume (predicted FEV1) expected
for a healthy individual of the same age, gender and
height (9, 10). Patients were considered pancreatic suf-
ficient according to fecal pancreatic elastase level or
evidence of malabsorption with a 24-h fecal fat mea-
surement. The study was approved by the Institutional
Review Board at CHB.
Genomic DNA was evaluated for the presence of CFTR
mutations (Genzyme, San Francisco, CA or Ambry
Genetics, Aliso Viejo, CA) as part of the clinical
evaluation. The entire gene was sequenced. None of
the subjects had more than one mutation within each
CFTR gene and no cis mutations were identified.
The non-p.F508del alleles of the p.F508del compound
heterozygotes (one allele is p.F508del and the other is a
known non-p.F508del CFTR mutation) were classified
as ‘mild’ if the biological classification score was class
IV or V, or ‘severe’ if the biological classification score
was class I, II or III. From henceforth, we refer to this
modified biological classification score as the biological
Next, the analysis was restricted to the non-
synonymous missense amino acid mutations. Grantham
scores were then calculated for each mutation. Grantham
scores are a measure of the chemical dissimilarity
between the native and the substituted amino acid and
are based on the amino acid substitution matrix of
Grantham (11). Higher Grantham scores are usually
predictive of severe mutations as defined by in vitro
assays (12). The SIFT score is correlated with whether
the mutation is at an evolutionarily conserved amino
acid residue. Mutations that occur in evolutionarily con-
served regions are hypothesized to be deleterious and
result in a more severe clinical phenotype (13). SIFT
score values greater than or equal to 0.05 are predicted
as tolerant (14). All non-synonymous missense muta-
tions in the study occurred at evolutionarily conserved
sites across the following eight vertebrate orthologs:
baboon, macaque, cow, sheep, rabbit, mouse, killifish,
Genotype-phenotype correlations in CF
Multivariate linear regression models adjusting for
age and sex using the biological classification, Grantham
and SIFT scores as covariates were used to predict
sweat chloride levels and predicted FEV1. Multivariate
logistic regression models using these covariates were
used to determine the probability of pancreatic insuffi-
ciency and risk of Pseudomonas infection in the past
year. To ensure robust results, we randomly permuted
the biological, Grantham, and SIFT scores 100,000
times to make 100,000 datasets and a permutation test
p-value was calculated.
Subjects and genotypes
The genotype distribution for the 435 subjects evaluated
is shown in Table 1. A total 24.8% (146) were
heterozygous for the p.F508del mutation. Of the 146
p.F508del heterozygotes, a known CFTR mutation was
present in 74.0% (108) of the subjects, and the analysis
was restricted to these 108 subjects. Twenty-five non-
p.F508del alleles were identified (Tables 2 and 3).
Table 1. Genetic distribution of cystic fibrosis (CF) cohort
One F508del allele and one
known non-F508del CFTR
One F508del allele and an
Two known non-F508del alleles
One known non-F508del allele
and one unknown allele
Two unknown alleles
0 25 (5.7)
aThe subgroup used for the statistical analysis.
Relationship between phenotypes
Predicted FEV1 has an approximately normal distri-
bution (Shapiro–Wilks p = 0.61). Although the pre-
dicted FEV1 data were normalized for age, duration
of disease (age) was strongly associated with worse
predicted FEV1 (p = 1.17 × 10−8) suggesting older
individuals tend to have worse lung function. Males
had higher average predicted FEV1 and lower aver-
age sweat chloride values than females, however, these
differences were not statistically significant (p = 0.14
and p = 0.33, respectively). Males were at lower risk
of infection over the past year relative to females
(OR = 0.44, p = 0.044). Older individuals tended to
have higher sweat chloride values (r = 0.41, p = 1.8 ×
10−5) and higher odds of Pseudomonas infection (p =
7.6 × 10−6). No significant correlation existed between
predicted FEV1 and sweat chloride levels (p = 0.81)
Table 2. CFTR
mutation classificationfor compound
NA, not applicable; SIFT, Sorting Intolerant from Tolerant.
aThe following mutations biological classification scores could
not be verified: 1898+G-A, 394delTT, D1270N, R352Q, and
bAhmed et al. (27).
cWilschanski et al. (28).
dWelsh and Smith 29).
eMcKone et al. (21).
fLoubieres et al. (30).
gMoskowiz et al. (31).
or between sweat chloride concentrations and infec-
tion status with Pseudomonas after adjusting for age
and gender (p = 0.29). These results suggest that the
sweat chloride level at diagnosis is not a useful marker
of lung function. Pseudomonas infection within the
past year was associated with lower predicted FEV1
(p = 1 × 10−4) suggesting that lung infections portend
worse lung function.
Biological classification score as a predictor of CF
Subjects with ‘mild’ mutations tend to have lower
sweat chloride levels than subjects with more ‘severe’
mutations (p < 0.001). This association persisted after
adjusting for age and sex (p < 0.001), demonstrating
that the in vitro CFTR function correlates with the in
vivo sweat chloride levels. There was no significant
association between mutation biological classification
and predicted FEV1on either univariate analysis (p =
Sebro et al.
Table 3. Biological classification scores for the CFTR mutations in cohort
Number infected with
aeruginosa in past year
deviation) Male (%)
Class I: no synthesis
Class II: block in processing
Class III: block in regulation
Class IV: altered conductance
Class V: reduced synthesis
0.37) or multivariate analysis (p = 0.98) (Table 4).
Seven of the eleven subjects with ‘mild’ CF geno-
types were pancreatic sufficient compared with 5 of
91 subjects with ‘severe’ CF genotypes (p < 0.001).
This association remained statistically significant after
adjusting for age and sex (p < 0.001). Subjects with
‘mild’ mutations had a lower probability of Pseu-
domonas infection in the previous year. Only 3 of
the 11 subjects with ‘mild’ CF genotypes had a Pseu-
domonas infection in the previous year compared to
60 out of 90 subjects with ‘severe’ CFTR mutations
(p < 0.001). This association remained significant after
adjusting for age and sex (p = 0.014).
Grantham and SIFT scores as predictors of CF phenotypes
in p.F508del compound heterozygotes with missense
Higher Grantham scores were associated with higher
sweat chloride levels (p < 0.001) and positively cor-
related with pancreatic insufficiency in the univari-
ate (p = 0.012) and multivariate analyses (p = 0.014).
Although there appeared to be an association between
higher Grantham scores and increased risk of Pseu-
domonas infection in the past year in univariate anal-
ysis (p = 0.027), this association disappeared in the
multivariate analysis (p = 0.082). There was no signifi-
cant association between Grantham score and predicted
FEV1on either univariate (p = 0.162) or multivariate
analyses (p = 0.278).
Univariate analysis showed that higher SIFT scores
were associated with lower sweat chloride levels in
both univariate (p < 0.001) and age and sex adjusted
analyses (p < 0.001). There was a positive correlation
between higher SIFT scores and pancreatic sufficiency
in both univariate (p = 0.009) and age and sex adjusted
analyses (p = 0.011). Subjects with higher SIFT scores
had a decreased risk of Pseudomonas infection in the
previous year in the univariate analysis (p = 0.046),
however, this association was marginal after adjusting
for age and sex (p = 0.058).
On the basis of biological classification score, more
‘severe’ mutations are associated with higher sweat
chloride levels and pancreatic insufficiency. Higher
Grantham scores and lower SIFT scores were associated
with pancreatic insufficiency and higher sweat chloride
levels amongst non-synonymous mutations. The risk
of Pseudomonas infection in the last year may be
associated with the CFTR mutation, with the risk
increased for subjects with more ‘severe’ mutations,
higher Grantham scores and lower SIFT scores. The
genetic scoring methods considered were not significant
predictors of predicted FEV1.
Table 4. Evaluation of CFTR mutation scoring methods to predict various components of the CF phenotype
Infected with P. aeruginosa
in past year (p-value)
score (mild vs severe)
Sex and age adjusted
Sex and age adjusted
Sex and age adjusted
4.0 × 10−7
1.4 × 10−8
4.8 × 10−5a
3.4 × 10−4a
2.1 × 10−7a
4.1 × 10−7b
1.4 × 10−5c
2.5 × 10−4b
CF, cystic fibrosis; SIFT, Sorting Intolerant from Tolerant.
ap-Values calculated from 100,000 permutations of mutation scores.
bp-Values calculated using Fisher’s exact test.
cp-Values obtained from logistic regression.
Genotype-phenotype correlations in CF
Our findings recapitulate previously published stud-
ies that discover no correlation between the pulmonary
function of CF subjects measured by predicted FEV1
and the CFTR mutation, and no correlation between
the sweat chloride level at age of diagnosis and the
predicted FEV1(15–19). The data support the in vitro
biological classification method as a means of classify-
ing the CFTR mutations because milder mutations have
been associated with in vivo pancreatic sufficiency and a
milder clinical phenotype (6, 20, 21). Urban et al. found
that higher Grantham scores and lower SIFT scores
were associated with worse in vitro function (12). An
analysis of SIFT, Panther and PolyPhen by Dorfman
et al. (22) showed that SIFT and Panther are able to
predict disease causing mutations, albeit without 100%
sensitivity and specificity. Dorfman et al. also showed a
positive association between disease severity and SIFT
scores, although the association was not significant.
These findings suggest that in silico tools capture only
some of the information in a mutation and should not
be used as clinical diagnostic tools. Finally, Kubesch
et al. asserted that CFTR genotype predicts the risk
of airway colonization with Pseudomonas (8), possibly
directly through its role as an epithelial receptor for this
microorganism (7). Murine models of CF have shown
these mice have increased susceptibility to colonization
with Pseudomonas (23). We find borderline evidence
to support this hypothesis.
The major clinical implication of our findings is that
sweat chloride levels, pancreatic function, and Pseu-
domonas infection risk appear to be associated with the
CFTR mutation. This suggests treatments that affect the
amount of functional CFTR are probably more likely to
affect these phenotypes than the pulmonary phenotype.
The pulmonary phenotypic heterogeneity in CF subjects
with the same genotype is probably due to modifier
genes, non-genetic factors or modifier genes interacting
with non-genetic factors.
There are a few study limitations. The cohort is a
clinical cohort and not a prospective cohort. Data was
collected retrospectively and all data collection was
performed blinded to the subjects’ genotypes, and there-
fore, not likely to affect the results. The power of the
study is limited because of the small sample size and
the small numbers of subjects with a given CFTR muta-
tion, although our results are very much in concert
with previously published reports. The in vivo function
of the membrane transporter may differ for a different
substrate or the membrane transporter may have a non-
transport function (12), therefore the biological classi-
fication of a mutation may not capture the complete in
vivo impact of the mutation. In addition to the trans-
port of diverse substrates (18), CFTR may have non-
transport functions as a receptor for Pseudomonas (16).
The p.R117H mutation has a variable penetrance as its
splicing efficiency is affected by the length of the poly-
T-tract in intron 8 (IVS8-5T, 7T and 9T), therefore the
genotyping scoring tools are probably inadequate for
evaluation of this mutation (24–26). Finally, although
we found no association between CFTR mutation and
predicted FEV1, further research should be performed
following pulmonary status over time, as a longitudi-
nal assessment of pulmonary function may prove to be
The diversity of lung disease in CF subjects may
not be related to variation in CFTR mutations, as
there is considerable phenotypic heterogeneity, even
in subjects with the same genotype or class of CFTR
mutation. Understanding the complex interplay between
mutational variants and phenotype using CF and other
monogenic diseases will prove to be important as
mutational variants for complex diseases are identified.
We are grateful to Dr Sudhir Kumar, who provided us with the
data on the evolutionarily conserved domains in the CFTR gene.
We are also grateful to Solandra Craig, Christopher Garcia and
the anonymous reviewers for the critical review of the manuscript.
R. S. was supported by a Howard Hughes Medical Institute Pre-
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